首页 | 本学科首页   官方微博 | 高级检索  
   检索      

TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究
引用本文:王淑君,张德蒙,李娜,王贝贝,谢红国,郭昕,白凤武.TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究[J].现代生物医学进展,2016,16(10):1801-1806.
作者姓名:王淑君  张德蒙  李娜  王贝贝  谢红国  郭昕  白凤武
作者单位:大连理工大学生命科学与技术学院;中国科学院大连化学物理研究所
基金项目:国家自然科学基金项目(51103157,81171555)
摘    要:目的:构建真核表达载体p IRES-EGFP-BMP-2,通过Turbo Fect转染得到表达BMP-2蛋白的CHO细胞系。方法:利用逆转录PCR方法扩增获得人的BMP-2基因c DNA,克隆入p MD18-T载体,经PCR、酶切和基因测序分析等方法鉴定重组质粒;将BMP-2连入p IRES-EGFP真核表达载体中,经限制性酶切和PCR扩增鉴定重组质粒。以壳聚糖和Turbo Fect分别作为基因载体转染CHO细胞,荧光显微镜检测分析转染结果;G418筛选富集转染阳性细胞。结果:成功的克隆得到了BMP-2基因,酶切鉴定成功构建了p IRES-EGFP-BMP-2质粒。与壳聚糖组相比,Turbo Fect用量为1:1时,细胞阳性率为(31.92±1.31)%,高于壳聚糖的细胞阳性率(6.33±1.53)%。目的基因与Turbo Fect比例为1:2时转染效率为(42.90±1.10)%高于1:1的(28.59±2.38)%和1:3的(37.52±2.14)%。细胞密度调节到5×103 cells/cm2阳性细胞率可达到(44.43±3.23)%。荧光检测可见荧光阳性细胞得到稳定传代。Western Blot检测可见BMP-2蛋白表达。结论:Turbo Fect成功的介导了p IRES-EGFP-BMP-2载体转染CHO细胞,建立了稳定表达BMP-2和EGFP的CHO细胞株。

关 键 词:骨形态发生蛋白2  转染  阳离子聚合物  壳聚糖  TurboFect

CHO Transfected by TurboFect-mediated Bone Morphogenetic Protein 2
Abstract:Objective:To construct the eukaryotic vector pIRES-EGFP-BMP-2 and get the BMP-2 expressed in CHO cells by cationic polymers.Methods:Human BMP-2 cDNA was amplified by RT- PCR, and inserted into cloning pMD18-T vector. The recombinant plasmid was identified by processes of PCR amplification, restriction expression vector, the vector was identified by enzyme digestion and PCR amplification. Chitosan and TurboFect were taken as carriers to transfect CHO cells, respectively. The transfection efficiency was analyzed with fluorescence microscope analysis results of transfection. Finally, the positive cells screened by G418.Results:The BMP-2 gene was been successfully cloned and the construction of pIRES-EGFP-BMP-2 was verified by restriction enzyme digestion. When the dosage of TurboFect is 1:1, fluorescent cell positive rate was(31.92± 1.31) %, which was higher than chitosan group (6.33± 1.53)%]. The fluorescent cell positive rate was (42.90± 1.10) %, when the dosage of TurboFect is 1:2, which was higher than (28.59± 2.38)%and (37.52± 2.14)%when the dosage of TurboFect is 1:1 and 1:3. The fluorescent cell positive rate could reach up to (44.43± 3.23) %by adjusting cell inoculum density to 5× 103 cells /cm2. After screened by G418 BMP-2 protein was stabled expressed in CHO cells.Conclusion:The pIRES-EGFP-BMP-2 has been successfully transfected into CHO cells by TurboFect. The study has established CHO cell lines stable expressed BMP-2 and EGFP.
Keywords:Bone morphogenetic protein-2  Transfection  Cationic polymers  Chitosan  TurboFect
本文献已被 CNKI 等数据库收录!
点击此处可从《现代生物医学进展》浏览原始摘要信息
点击此处可从《现代生物医学进展》下载免费的PDF全文
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号